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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

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Published on: July 2, 2010

Protocol for mapping RNA G-quadruplex for chromatin-bound RNA using d-rG4-seq.

Yong Woo Lee¹, Vered Levy¹, Jeannie T Lee¹

¹Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.

|December 7, 2024

Summary

This study introduces d-rG4-seq, a method to map RNA G-quadruplexes (rG4) in chromatin-bound RNA. The protocol helps identify in vivo rG4 structures by analyzing their modification and folding properties.

Keywords:

Gene Expression Genomics Molecular Biology

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Last Updated: Jun 12, 2025

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

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Published on: July 2, 2010

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

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High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Published on: October 5, 2018

Area of Science:

Molecular Biology
Genomics
Epigenetics

Background:

RNA G-quadruplexes (rG4) are crucial non-coding regulatory elements.
Understanding the in vivo landscape of rG4 structures is essential for deciphering their regulatory roles.
Chromatin-bound RNA plays a significant role in epigenetic regulation.

Purpose of the Study:

To present a detailed protocol for d-rG4-seq, a technique for mapping RNA G-quadruplexes (rG4) in chromatin-bound RNA.
To enable the identification of in vivo rG4 structures based on their biophysical properties.
To facilitate the comparison of in vitro and in vivo rG4 profiles and isolate chromatin-associated RNA.

Main Methods:

d-rG4-seq protocol development.
Differential sensitivity of rG4 to dimethyl sulfate (DMS) modification.
Assessment of rG4 folding in the presence of monovalent cations (K+ vs. Li+).
Analysis of reverse transcriptase (RT) readthrough for folded rG4 structures.
Isolation of RNA from chromatin-bound fractions.
Comparison of in vitro and in vivo rG4 profiles.

Main Results:

A robust protocol for mapping chromatin-bound RNA G-quadruplexes (rG4) is established.
The method allows for the identification of in vivo rG4 structures.
Differential chemical probing and enzymatic readthrough reveal structural characteristics of rG4.
Isolation of chromatin-associated RNA enriches for epigenetic regulators.

Conclusions:

The d-rG4-seq protocol provides a powerful tool for studying the functional landscape of RNA G-quadruplexes in their native chromatin context.
This technique aids in understanding the role of rG4 in epigenetic regulation.
The protocol facilitates the comparison of RNA G-quadruplex structures in vitro and in vivo.